1. Field of the Invention
This invention relates to a method of investigating materials using x-rays and to apparatus therefor. More particularly, the invention relates to investigating biological specimens under ambient conditions of the environment by means of an x-ray microscope (XM). In a preferred embodiment, a novel scanning x-ray microscope (SXM) is provided and utilized in the method of the invention.
From the time of the discovery of x-ray radiation, x-rays have been utilized in many different ways for the investigation of materials including biological materials. Microscopes have been of great importance in the investigation of both biological and other materials. Optical microscopes are limited in resolution by the wavelength of the photons in a beam of visible light. The electron microscope (EM) was developed using the electromagnetic lens to focus the electrons. Electrons can have extremely short wavelengths which enables the EM to have very high magnification and greatly improved resolution compared with optical microscopes. Electrons are also employed in a scanning electron microscope (SEM), which operates in a different way from the EM. An SEM employs a beam of electrons focused to a spot having a diameter of several nm which is scanned over the surface of a sample in a pattern generating secondary electrons which are detected and amplified for each pixel to collectively compose a picture of the surface of the sample. Compared with an optical microscope, a far better resolution and depth of field is obtained.
However, investigation of materials with an EM or SEM requires examining the specimens in an evacuated environment for the passage of electrons, as well as treatment of the specimens. For investigation with an EM, a very thin specimen must be prepared and stained with heavy metals. The sample for a SEM requires coating with heavy metals.
2. Description of Related Art
A scanning x-ray microscope (SEM) is disclosed in U.S. Pat. No. 4,317,036 granted on Feb. 23, 1982 to Chia-Gee Wang. This patent discloses that prior attempts to construct x-ray microscopes were based on equipping electron microscopes with x-ray detectors and that such a system can function as an x-ray microscope with scanning electron detection. Among the factors stated to cause the prior XM to become complex and expensive was the need for a vacuum housing for the electron beam and the specimen. The invention of this patent was directed towards a more simple system in which a beam of x-rays was attempted to be focused onto a small spot for scanning the beam over a specimen, without the use of vacuum. However, it has been found that the necessary precise focusing of the x-ray beam is complex and difficult in many cases. Accordingly, there is a need in the art for a system which avoids the difficulty of focusing an x-ray beam, while providing a means of analyzing materials or specimens under normal conditions not involving the application of a vacuum to the specimens.
Many types of equipment have been developed to utilize x-rays for investigation or analysis of materials. One such type is an x-ray analyzer, electron-micro probe, generally described in "Van Nostrand's Scientific Encyclopedia", Sixth Edition, pages 3041-3044, the disclosure of which is incorporated herein by reference. This instrument is mainly used for metal-lurgical studies but properly prepared biological specimens may also be analyzed. An optical microscope is used to identify a point of interest on the specimen to be analyzed. An electron beam is focused on such point on the specimen and resulting x-rays are detected and processed to provide quantitative data. In the instrument of this reference, the electron beam is focused directly onto the specimen, both the beam and the specimen being under vacuum. The reference further discloses the production of electron beams and detectors for detection of x-rays.
The field is generally reviewed in "X-ray Microscopy", Proceedings of the International Symposium, Gottingen, Fed. Rep. of Germany, Sept. 14-16, 1983, edited by G. Schmal and D. Rudolph, Springer-Verlag, 1984, the contents being given on pages vii-ix.
X-rays have also been used for the detection of atoms in biological specimens using monochromatic x-rays, as disclosed in U.S. Pat. No. 4,239,966 issued on Dec. 16, 1980 and U.S. Pat. No. 4,382,181 issued on May 3, 1983, both issued to Chia-Gee Wang.
An article in SCIENCE, vol. 237, Aug. 14, 1987, pages 723-724 discusses current research in scanning x-ray microscopy. The article states that biological material in an aqueous environment has been imaged by scanning x-ray microscopy employing a synchrotron light source. X-rays from the light source are focused with use of a Fresnel zone plate. The system is described as not being perfected in view of lengthy time to record images so that dynamic processes cannot be studied and because of noise in the images. Future attempts are to involve brighter soft (long wavelength) x-ray sources, but the article states that it is yet to be demonstrated that cell structures will always survive this soft and intense x-ray dose. Also disclosed in the article are other prior techniques such as contact x-ray micrography in which shadowgraphs of samples illuminated by an x-ray beam are recorded by lithographic means and a transmission electron microscope is used to magnify the image. Prior work has also involved the x-ray analog of an optical microscope. Both the contact method and the optical analog method utilize an intense beam of soft x-rays from a synchrotron light source. The use of a scanning instrument is suggested because of the potential to employ a lower radiation dose and to follow changes within living cells, but the disadvantage of scanning is explained as involving the need to use a very bright x-ray source.